The mitochondrial proteome consists of about 1500 proteins, the majority of which are encoded in nuclear DNA, synthesized on cytosolic ribosomes, and post-translationally targeted to the organelle. The TIM23 complex of the mitochondrial inner membrane mediates the import of most of these proteins. The initial stages of protein import involve a receptor complex composed of the soluble domains of the Tim23 and Tim50 subunits. This receptor not only coordinates the recognition of mitochondria-targeted polypeptides, but also aids in the maintenance of the inner membrane potential and in the processing of mitochondrial enzymes. Despite its relevance in organelle biogenesis and human disease, the molecular mechanisms by which the TIM23 receptor regulates the early steps of protein import are poorly understood. Using innovations in model membrane constructions and an experimental strategy that combines advanced fluorescence spectroscopy, molecular modeling, and structural biology techniques, this research plan will investigate the network of protein and lipid interactions that underpin the operation of the TIM23 receptor. In Aim 1, we will analyze the dynamic associations between the Tim50 and Tim23 subunits under different conditions. With both in organello systems and soluble nanoscale lipid membrane models, we will employ site-specific crosslinking and fluorescence measurements to elucidate how the associations of these receptor subunits are regulated by substrate, membrane potential, and particular lipids. These results will resolve the molecular mechanisms by which the TIM23 receptor makes specific contact with targeted substrates and how the receptor gates the translocon channel for import. In Aim 2, we will investigate the lipid interactions of the receptor subunits. Based on our preliminary work indicating that specific sites on Tim23 and Tim50 bind to bilayers containing the mitochondria- specific lipid cardiolipin, we will analyze how interactio with this lipid may induce structural changes in these proteins and what implications this has for operation of the receptor. These results will define a novel paradigm for the role of protein-lipid interactions in regulating protein import, and offer insights into the molecular basis of heritable diseases linked to defects in mitochondrial lipid biogenesis. In Aim 3, we will employ a host of independent approaches to determine the high-resolution structure of the assembled Tim23-Tim50 receptor complex. The conformational changes of these cognate binding partners that occur upon formation of the receptor will be interpreted in the context of their interactions with lipids and substrate. This work will rigorously test and refine our unifying working model for the initial steps of TIM23 complex-mediated protein import. From a broader perspective, this research will give many critical insights into the regulation of cellular protein trafficking and mitochondrial biogenesis.